This invention relates to the cytosolic translocation factor complex (CTL) responsible for the translocation of the catalytic domain of diphtheria toxin from the lumen of endosomes to the cytosol.
Diphtheria toxin (DT) (58 kDa) is a typical single chain AB toxin composed of three functional domains: the amino terminal catalytic (C) domain corresponds to fragment A (21 kDa), and the transmembrane (T) and carboxy terminal receptor binding (R) domains comprise fragment B (37 kDa) of the toxin (Choe et al., Nature 357: 216-22, 1992). A disulfide bond between Cys186 and Cys201 subtends a protease sensitive loop and connects fragment A with fragment B. Furin mediated cleavage within this loop and retention of the disulfide bond have been shown to be pre-requisites for intoxication of eukaryotic cells (Tsuneoka et al., J. Biol. Chem. 268:26461-5, 1993; Ariansen et al., Biochem. 32:83-90, 1993). Substitution of the native R domain with human interleukin-2 (IL-2) has resulted in the formation of a fusion protein toxin, DAB389IL-2, whose cytotoxic action is specifically targeted only to cells expressing the high affinity IL-2 receptors (Bacha et al., J. Exp. Med. 167:612-622, 1988; Waters et al., Eur. J. Immunol. 20:785-91, 1990; Ratts and vanderSpek, Diphtheria Toxin: Structure Function and its Clinical Applications. In Chimeric Toxins, H. Lorberboum-Galski, P. Lazarovici, eds., Taylor and Francis, London, New York. p. 14-36, 2002).
The intoxication of eukaryotic cells by diphtheria toxin follows an ordered series of interactions between the toxin and the cell which leads to inhibition of protein synthesis and cell death (Love and Murphy, Gram-Positive Pathogens, American Society for Microbiology, Washington, D.C., V. A. Fischetti, J. Rood Ed. pp. 573-582, 2000). Biochemical, genetic and X-ray crystallographic analysis of the toxin has shown the protein to be composed of three distinct domains: an N-terminal catalytic domain (C-domain), a central transmembrane domain (T-domain), and the C-terminal receptor binding domain (R-domain). The intoxication process is initiated by the binding of the toxin to its cell surface receptor, a heparin binding epidermal growth factor-like precursor and CD9. Once bound to its receptor, the toxin is internalized by receptor-mediated endocytosis into an early endsosomal compartment (Moya et al., J. Cell. Biol., 101:548, 1985). Upon acidification of the endosomal lumen by vesicular (v)-ATPase, the T-domain undergoes a conformational change and spontaneously inserts into the vesicle membrane forming an 18-22 Å pore or channel (Kagan et al., Proc. Natl. Acad. Sci., USA, 78:4950, 1981; Donovan et al., Proc. Natl. Acad. Sci., USA, 78:172, 1981). The C-domain, in a fully denatured form, is then specifically thread through this channel and released into the cytosol. Once the C-domain is refolded into an active conformation it catalyzes the NAD+-dependent ADP-ribosylation of elongation factor 2 (EF-2), causing irreversible inhibition of protein synthesis and death of the cell by apoptosis (Pappenheimer, Annu. Rev. Biochem., 46:69, 1977; Kochi and Collier, Exp. Cell. Res., 208:296, 1993).
The requirements for C-domain translocation of diphtheria toxin across endosomal membranes have been partially defined in PCT patent application publication number WO2005014798. In general, non-toxic mutants of diphtheria have fallen into one of two categories: point mutants that no longer catalyze the NAD+-dependent ADP-ribosylation of elongation factor 2 (e.g., CRM197; see Uchida, J. Biol. Chem. 248:3838, 1973) and premature chain termination mutants that are no longer capable of binding to the eukaryotic cell surface receptor for the toxin (e.g., CRM45; see Uchida, vide supra). The construction, isolation, and properties of a series of site-directed mutations in transmembrane helix 1 of DAB389IL-2 have been previously reported (vanderSpek et al., Protein Eng. 7:985, 1994). In this series, the non-toxic DAB389(L221E)IL-2 mutant was of particular interest since it was both ADP-ribosyltransferase positive and bound to the targeted high affinity IL-2 receptor with an affinity equal to that of the wild type fusion protein. It was also found that DAB389IL-2 binds with greater affinity to its receptor compared to native DT. Therefore, this fusion protein toxin has proven to be an effective probe for studying internalization of the C-domain by target cells (Williams et al., J. Biol. Chem. 265:11885-9, 1990).
While much is known about the mechanisms of receptor binding and receptor mediated endocytosis of native DT and the DT-related fusion proteins, less is known about the precise molecular mechanisms of C-domain translocation across the endosomal membrane and its release into the cytosol.